Most biological processes that require both high fidelity and complex cellular regulation are carried out by elaborate multicomponent machines. The spliceosome, which removes introns from pre-mRNAs, is a striking example of this kind machine. Five small nuclear RNAs (U1, U2, U4, U5 and U6) and a minimum of fifty distinct protein components are present in the spliceosome. The extreme levels of specificity required for splicing are achieved through a combination of the dynamic spliceosome assembly process and the formation of complex networks of RNA-RNA, RNA-protein and protein-protein interactions. The long-term goal of our research is to achieve a detailed understanding of the mechanisms of spliceosome assembly. To accomplish this goal it is necessary to identify all of the functional components of the spliceosome complexes and develop purified systems for reconstituting them. In this proposal we plan to use two complementary approaches towards these goals. In the first approach we plan to establish a procedure for isolating highly purified, functional splicesomal complexes (Aim 1). Detailed analyses of the compositions of these complexes and structure-function studies should provide important insights into the mechanisms of spliceosome assembly. In parallel we plan to establish a purified system for reconstituting the early steps of spliceosome assembly (Aim 2). Together, these approaches will provide the first opportunity to directly test specific models for the mechanisms of early spliceosome assembly. In particular, we plan to investigate the early roles of the highly conserved multicomponent splicing factors, SF3a and SF3b, both of which interact with pre-mRNA near or at the catalytic core of the spliceosome. A key objective is to assemble SF3a and SF3b from recombinant subunits and then use these complexes, and ultimately mutant derivatives of them, in the reconstitution system. Through order of addition studies, analysis of mutant pre-mRNAs, and comparison of purified native complexes to our reconstituted complexes, we anticipate making significant progress in elucidating the early steps in spliceosome assembly. Another central focus of our work is on the second catalytic step of the splicing reaction (Aim 3). We plan to use both systematic analysis of mutant pre-mRNAs and characterization of proteins (hPrp16, hPrp17 and hSlu7) specifically involved in catalytic step II in order to investigate the step II mechanism.